Eukaryotes have evolved mechanisms for maintaining energy homeostasis in the face of changes in nutrient availability and energy expenditure. AMP-activated protein kinase (AMPK) is a conserved sensor of cellular energy status, acting as a critical node in signaling networks controlling cellular metabolism. Suppression of AMPK activity has been implicated in insulin resistance, obesity, cancer, and heart disease, and a major clinical drug for treating type II diabetes is an AMPK activator. AMPK is activated by cellular energy stress, and its subsequent phosphorylation of multiple target proteins serves to increase catabolism and decrease energy consumption to maintain energy balance. While several key phosphorylation targets of AMPK are known, it is likely that there are many additional substrates that remain to be discovered. These studies will focus on Snf1, the yeast ortholog of AMPK, which has long served as an important model for studying AMPK regulation and function. We propose to identify a large number of novel Snf1 substrates using emerging targeted phosphoproteomics methodology. Through motif-based analysis of shotgun phosphoproteomics data, we have identified approximately 100 potential substrates of Snf1. We will develop assays for relative quantification of the phosphorylation state of these substrates in cell extracts using a targeted mass spectrometry approach. Sites that decrease in abundance following chemical-genetic inhibition of Snf1 are considered to be dependent on the kinase in vivo. We will then use a novel genetic method to establish which sites are directly phosphorylated by Snf1. We have generated a Snf1 mutant by structure-based design that exchanges its phosphoacceptor residue preference from Ser to Thr. By introducing compensating mutations at the phosphorylation site of substrates, we can restore phosphorylation by mutant Snf1. The ability to generate functional re- engineered kinase-substrate pairs in vivo provides strong evidence of direct phosphorylation. We will characterize Snf1-dependent phosphorylation networks controlling glycerolipid metabolism and autophagy through detailed functional analysis of novel direct substrates. Substrates predicted to be conserved to humans will be examined for regulation by AMPK in mammalian cells. These studies will provide fundamental insight into mechanisms by which Snf1 and human AMPK control cellular metabolism in response to changes in nutrient availability. In addition, the methodology used for these studies should be applicable other kinases as well, providing general tools for elucidating phosphorylation-dependent signaling networks in eukaryotes.